Hypoxia plays an important role in physiology, pathophysiology and cancer. Recent examples include: hormonal control of tissue oxygenation (Badger et al., Urol Int, 76: 264-268, 2006); bone remodeling (Dodd et al., Am. J. Physiol. Renal. Physiol 277: C598-602, 1999); embryogenesis (Nanka et al., Dev Dyn, 235: 723-733, 2006); teratogenesis (Danielsson et al., Birth Defects Res A Clin Mol Teratol 73: 146-153, 2005); optic nerve ischemia (Danylkova et al., Brain Res 1096: 20-29, 2006); ischemic heart disease (Cheema, et al., J Am Coll Cardiol 47: 1067-1075, 2006); inflammatory disease including arthritis (Peters et al., Arthritis Rheum 50: 291-296, 2004); wound healing (Albina et al., Am J Physiol Cell Physiol 281: C1971-1977, 2001); ischemic kidney disease (Villanueva et al., Am J Physiol Regul Integr Comp Physiol 290: R861-870, 2006); cirrhotic liver disease (Jeong et al., Liver Int 24: 658-668, 2004); lung disease (Morani et al., Proc Natl Acad Sci USA 103: 7165-7169, 2006); alcohol induced pancreatic disease (McKim et al., Arch Biochem Biophys 417: 34-43, 2003); thymic disease (Hale et al., Am J Physiol Heart Circ Physiol 282: H1467-1477, 2002); obstructive disease of urogenital organs (Damaser et al., J Appl Physiol 98: 1884-1890, 2005; Ghafar et al., J Urol 167: 1508-1512, 2002); and, in cancer prognoses (Carnell et al., Int J Radiat Oncol Biol Phys 65: 91-99, 2006; Kaanders et al., Cancer Res 62: 7066-7074, 2002).
Two categories of hypoxia are currently recognized in solid tissues: diffusion-limited chronic hypoxia and perfusion-limited acute or fluctuating hypoxia. In addition to their impact on local radiation control in tumors, acute and chronic hypoxia are believed to contribute to an overall poor prognosis for cancer patients by inducing hypoxia-induced angiogenesis, migration, and invasion factors that increase overall tumor aggressiveness independent of treatment protocol (Vaupel et al., Semin. Oncol. 28:29-35, 2001). Chronic hypoxia is a natural feature of normal tissues such as liver and kidney and is not a pathophysiological condition. However, uncontrolled fluctuations in hypoxia contribute to hypoxia-reperfusion injury by creating reactive oxygen species in normal tissue (Thurman et al., J. Gastroenterol. Hepatol. 13(Suppl):S39-50, 1998).
Chronic hypoxia arises at the distal end of oxygen gradients created by oxygen consumption in cells close to blood vessels compounded, in the case of tumors, by deficiencies in local oxygen supply arising from longitudinal gradients of pO2 in tumor vascular trees (Dewhirst et al., Int. J. Radiat. Oncol. Biol. Phys. 42:723-726, 1998). Thomlinson and Gray first deduced that regions of chronic hypoxia exist in human tumors and proposed that these regions contribute to tumor radiation resistance (Thomlinson and Gray, Br. J. Cancer 9:539-549, 1955).
Acute hypoxia, in contrast to chronic hypoxia with static, metabolically controlled pO2 gradients, is associated with fluctuating pO2 that results from blood flow instabilities which, in the case of tumors, is created by transient vascular occlusion (Dewhirst et al., supra). It has been proposed that acutely hypoxic tumor cells, being proliferative, might be more therapeutically relevant (Wouters et al., Radiat. Res. 147:541-550, 1997) than quiescent, chronically hypoxic cells (Kennedy et al., supra; Varia et al., Gynecol. Oncol. 71:270-277, 1998). In normal tissues, fluctuating hypoxia is associated with hypoxia-reperfusion injury such as alcohol-induced liver disease (Arteel et al., Am. J. Physiol. 271:G494-500, 1996); alcohol-induced pancreatitis (McKim et al., Arch. Biochem. Biophys. 417:34-43, 2003); and, chemotherapy-induced kidney disease (Zhong et al., Am. J. Physiol. 275:F595-604, 1998).
Immunohistochemical hypoxia markers have been used to clearly visualize oxygen gradients in human tumors (Raleigh et al., Br. J. Cancer 56:395-400, 1987; Cline et al., Br. J. Cancer 62:925-931, 1990; Kennedy et al., Int. J. Radiat. Oncol. Biol. Phys. 37:897-905, 1997; and U.S. Pat. No. 5,086,068) and were subsequently used to demonstrate that cellular hypoxia was prognostic for outcome in head and neck cancer (Kaanders et al., Cancer Res. 62:7066-7074, 2002). One of these markers, the HCl salt of the weakly basic 2-nitroimidazole, pimonidazole (1-(2-hydroxy-3-piperidinopropyl)-2-nitroimidazole, pKa=8.7), has been used to measure hypoxia by immunochemical means (U.S. Pat. No. 5,674,693; U.S. Pat. No. 5,086,068). Immunohistochemical analyses are useful for relating cellular hypoxia to other physiological factors such as oxygen-regulated protein expression, vasculature, necrosis, and cellular differentiation, but because they require biopsy tissue, they are invasive, subject to sampling error, unsuitable for routine clinical studies of normal tissue hypoxia, and are less desirable for following changes in human tissue hypoxia because of the inconvenience and discomfort associated with sequential biopsy.
In 1976, Varghese et al. showed that nitroheterocyclic compounds are reductively activated and covalently bound to hypoxic mammalian cells (Varghese et al., Cancer Res. 36:3761-3765, 1976). The addition of the first electron in the cascade of electrons from cellular electron transfer systems that bioreductively activate 2-nitroimidazole hypoxia markers is reversible by molecular oxygen whereby the binding of the markers becomes an indirect measure of tissue hypoxia. In 1981, Chapman et al. demonstrated that the oxygen dependence of binding was in the range of pO2 that rendered tissues resistant to radiation damage (Chapman et al., Br. J. Cancer 43:546-550, 1981). Following the discoveries of Varghese et al. and Chapman et al., attempts were made to translate them into clinically useful techniques for measuring tissue hypoxia. Invasive techniques included autoradiography and scintillation counting of radioactively-labeled 2-nitroimidazoles (Urtasun et al., Br. J. Cancer 54:453-457, 1986); antibody based immunohistochemistry (Raleigh et al., supra; Cline et al., supra; and U.S. Pat. No. 5,086,068); antibody based enzyme linked immunosorbent assay (Raleigh et al., Br. J. Cancer 69:66-71, 1994); and, antibody-based, flow cytometry (Olive et al., Acta. Oncol. 40:917-923, 2001).
Early non-invasive techniques for measuring tissue hypoxia included single photon emission tomography (SPECT; Urtasun et al., Br. J. Cancer Suppl. 27:S209-12, 1996; Iyer et al., Br. J. Cancer 78:163-9, 1998); nuclear medicine (Ballinger et al., J. Nucl. Med. 37:1023-31, 1996; Strauss et al., J. Nucl. Cardiol. 2:437-45, 1995); [19F]magnetic resonance spectroscopy (Raleigh et al., Int. J. Radiat. Oncol. Biol. Phys. 12:1243-5, 1986; Jin et al., Int. J. Radiat. Biol. 58:1025-34, 1990); and positron emission tomography with 18F-fluoromisonidazole ([18F]FMISO; Rasey et al., Int. J. Radiat. Oncol. Biol. Phys. 17:985-991, 1989). A number of reagents were invented for the purpose of improving upon [18F]fluoromisidazole ([18F]MISO). These included [18F]fluoroetanidazole ([18F]FETA; Rasey et al., J. Nucl. Med. 40:1072-1079, 1999); [18F]fluoroerythronitroimidazole ([18F]FETNIM; Yang et al., Radiology 194:795-800, 1995; Wallace et al., U.S. Pat. No. 5,728,843); [18F]2-(2-nitro-1H-imidazol-1-yl)-N-(3-fluoropropyl)-acetamide([18F]EF1; Evans et al., J. Nucl. Med. 41:327-336, 2000; Koch et al., U.S. Patent Appln. Publn. No. 2005/0026974 A1); [18F]2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3-pentafluoropropyl)-acetamide ([18F]EF5; Ziemer et al., Eur. J. Nucl. Med. Mol. Imaging 30:259-266, 2003; Dobler et al. U.S. Patent Appln. Publn. No. 2006/0159618 A1); [18F]fluoroazomycinarabinofuranoside ([18F] FAZA; Sorger et al., Nucl. Med. Biol. 30:317-326, 2003); 4-bromo-1-(3-[18F]fluoropropyl)-2-nitroimidazole (4-Br-[18F]FPN); and 1-(3-[18F]fluoropropyl)-2-nitroimidazole ([18F]FPN; Yamamoto et al., Biol. Pharm. Bull. 25:616-621). Marginal improvements in tumor to normal tissue ratios, decreased liver uptake, and decreased circulating metabolites were achieved compared to [18F]FMISO.
PET measurement of tissue hypoxia with [18F] labeled 2-nitroimidazoles involves four more or less, independent processes: (1) fixed, rapid radioactive decay of [18F] (t1/2=109.8 minutes) attached to hypoxia marker adducts. This constitutes a rapidly decaying hypoxia signal against a dynamic background of (2) wash in and wash out of unmetabolized [18F]hypoxia marker molecules; (3) build up and catabolism of [18F] hypoxia marker protein adducts; and, (4) build up and wash out of [18F] small molecule metabolites of hypoxia markers that include cysteine and glutathione adducts and hydrolytic fragmentation products of the markers (Raleigh and Liu, Int. J. Radiat. Oncol. Biol. Phys. 10:1337-1340, 1984). Approximately 80% of bioreductively activated 2-nitroimidazole hypoxia markers are fragmented by hydrolysis. Fragmentation produces non-binding [18F] metabolites that make a major contribution to background noise but add nothing to the hypoxia signal. Approximately 20% of bioreductively activated 2-nitroimidazole hypoxia markers produce the hypoxia signal—10% from adducts with proteins and 10% from small, thiol containing compounds like glutathione (Raleigh and Koch, Biochem. Pharmacol. 40:2457-2464, 1990). Except for the absence of signal loss due to radioactive decay, non-invasive [19]MRS and [19F]MRI are subject to the same signal-to-noise considerations as [18]PET.
Mathematical models have been designed to isolate the hypoxia signal (protein and glutathione adducts) from background noise (unbound hypoxia marker and its non-binding metabolites), but kinetic data for the concurrent dynamic processes associated with hypoxia marker metabolism are essentially impossible to obtain on a patient-by-patient basis and PET investigators have adopted a simpler concept of fractional hypoxic tumor volume which is the proportion of tumor area (pixels) that possess a tumor-to-blood radioactivity ratio ≧1.4 at a fixed time of 2-3 hours post injection (Koh et al., Int. J. Radiat. Oncol. Biol. Phys. 33:391-398, 1995; Couturier et al., Eur. J. Nucl. Med. Mol. Imaging 31:1182-1206, 2004).
Early studies with 2-nitroimidazole compounds such as [18F]F-MISO (Rasey et al., Int. J. Radiat. Oncol. Biol. Phys. 17:985-991, 1989) and [19F]CCI-103F (Raleigh et al., Int. J. Radiat. Oncol. Biol. Phys. 12:1243-5, 1986) established the potential of [18F]PET and [19]MRS for measuring tissue hypoxia non-invasively, but there remains a need for reagents that improve sensitivity and specificity by improving signal-to-noise limitations for both chronic and acute hypoxia.